Basic strategy: consecutive coupling of mononucleotide building blocks
Chemical requirements:
- protection of reactive groups (a.o. on the nucleotide bases)
protection of 5’-end at one side and 3’-end at other side
- protections stable under conditions of the coupling reaction
- protections sufficiently labile to allow deblocking without strand cleavage
(and selective, if not all protections should be undone)
- extremely high yield : => theoretical yield versus number of cycles
(nb. there are several steps and substeps per cycle)

Theoretical yield resulting from a number of reaction cycles, at indicated efficiencies.
---------------------------------------------------
Number of cycles 90% 95% 99% 99.9%
80
90
100
40
50
60
70
5
10
20
30
59.0
34.9
12.2
4.2
1.5
0.5
0.2
<0.1
-
-
-
77.4
59.9
35.8
21.5
12.9
7.7
4.6
2.8
1.7
1.0
0.6
95.1
90.4
81.8
74.0
66.9
60.5
54.7
49.5
44.8
40.5
36.6
99.5
99.0
98.0
97.0
96.1
95.1
94.2
93.2
92.3
91.4
90.5

Chemistry
- phosphodiester : pioneering work by Gobind Khorana's group in the 50's and 60's (tri- and tetramers, linked together enzymatically, leading to elucidating the genetic code)
- phosphotriester : later in the '60's : protection of the 2 nd acidic group in the phosphate (by a cyanoethyl group ; by two independent groups, of R. Letsinger and C. Reese, respectively)
- 1970's : development of phosphite and phosphoramidite chemistries :
= "phosphotriester methods" with an initial 3-valent phosphorus
(reaction with the nucleoside to a phosphotriester intermediate)
- phosphoramidite : e.g. X=N(iPr)2
- phosphite : e.g. X=Cl (labs. of R.Letsinger and of M.Caruthers)
- phosphonate : based on initial work in the 1950's by A.Todd's group and
're-invented' and adapted in the 1980's to an efficient oligonucleotide synthesis chemistry
- one OH of the phosphate is replaced by an H
(in general phosphonate chemistry, the alternative P-H may also be a P-C linkage)

Chemistry
The phosphite and phosphoramidite methods require an oxidation step to phosphate at each cycle. The oxidation of phosphonate is done in a single reaction step at the global end of the synthesis (i.o.w. the oligonucleotide can also be stably isolated as a phosphonate oligonucleotide) .
Many variations are possible with respect to reagents and (requirement of) modifications.
The exocyclic NH
2 groups of the bases (A,C, G) are usually protected, although this is not always strictly necessary (but then may need additional reaction steps or alternative coupling agents and solvent systems).
Apart from the standard oligonucleotide synthesis, special substrates may be used at either the first or the final coupling step, to produce an oligonucleotide with a particular modification at either the 3' or 5' end, respectively. Such modifications (e.g. -NH
2
, -SH, etc.) may then be used for further derivatisations, or have specific functional properties
(such as fluorophore, quencher, biotin, etc.)
Extra steps may also be used during the cycles (see further) to produce e.g. phosphorothiate- or -selenoate- or -amidate-oligonucleotide variants.

phosphotriëster method phosphitetriëster method phosphoramidite method phosphonate method
DMT = dimethoxytrityl R = OHprotecting group R’ = P-protecting groep

adenosine and cytidine : benzoyl protected guanosine : isobutyryl protected thymidine does not need a protecting group
N m
N m
: nucleotide base with protecting group
DMT : dimethoxytrityl group
CPG : controlled pore glass

In practice
immobilisation of the 3’-end on a solid support mostly used : CPG (controlled pore glass) and MPPS (macroporous polystyrene) e.g. CPG pore sizes of 50, 100, 150, 200, and 300 nm are used for synthesis of about 50, 80, 100, 150, and 200-mer oligonucleotides, respectively.
- addition/removal of reagent is readily carried out
- simple washing procedures between the steps
- automatable
direction of synthesis : towards the 5’-end
- blocking of termini
blocking of the 5’-end with a dimethoxytrityl (DMT) moiety
3’-end bound to the solid support via a “linker”

START
1
1 4 nb. sulfurisation can be done instead of oxidation to prepare oligonucleotide phosphorothioates. Capping should then be done after sulfurisation. (This is also advisable with oxidation, according to some protocols.)
3
2
5'-OH nucleosidenucleotide product
(after start of 2 nd cycle) phosphoramidite reagent protected base

1. Detritylation : the trityl group is acid labile : cleavage by trichloroacetic acid (TCA)
2. Phosphoramidite :
- Di-isopropylamino group on a 3-valent phosphorus
- Very stable, but becomes very reactive with the activator tetrazole (CN
4
H
2
)
b
-cyanoethyl protecting group on the 3‘OH (can be removed by a.o. NH
3
)
3. 'Capping' blocks all unreacted ends (may be up to 2%) permanently : acetic anhydride + 1-methylimidazol = acetylating agent (in large excess)
=> elimination of incomplete reaction products from participation in the subsequent cycles (nb. the phosphoramidite reagents are expensive ; not to be used in excess !)
4. oxidation to 5-valent phosphorus : with iodine as weak oxidizing agent in a basic solution of tetrahydrofuran in water as oxygen donor.
3-valent phosphite 5-valent phosphotriester

Example : the phosphoramidite synthesis cycle :
4 major steps : (many substeps)
1- detritylation of the 5'-end
2- the coupling reaction
3- ' capping ‘ : blocking all non-reacted (5’) ends
(irreversibly)
4- oxidation to 5-valent phosphorus: phosphotriester : I
2 as a weak oxidant each step requires usually a number of manipulations ( substeps : addition of reagents, reaction time, washing, etc.
) reagent : e.g. guanosine cyanoethyl phosphoramidite

At the end of the final cycle :
Treatment with concentrated ammonia to
- cleaves the binding with the support
- hydrolyses the bonds of the protecting groups on the bases
- cleaves the phosphate-protecting cyanoethyl
The last trityl group (at the 5‘ end) can be removed together with or after the other deprotections
If after the other deprotections : with 80% acetic acid in water ;
This strategy allows for an extra chromatographic purification step (as a less polar DMT-oligonucleotide) prior to removal of the DMT.

Survey of basic steps in H-phosphonate oligonucleotide synthesis
S
8
= cyclooctasulfur, one of the many allotropes of sulfur

1986 : oligonucleotide synthesis on solid phase using 2'-deoxy and ribonucleoside H-phosphonate monoesters as synthons.
1) acid-mediated removal of the 5'-dimethoxytrityl protecting group,
2) condensation is carried out by addition of an appropriately protected 2'-deoxy or ribonucleoside H-phosphonate monoester (20) and an activating agent (e.g. pivaloyl chloride or adamantoyl chloride) . Reaction of the acid chloride with the H-phosphonate monoester produces the corresponding mixed phosphonocarboxylic anhydride. This mixed anhydride undergoes a nucleophilic attack at the phosphorous by the 5'- hydroxyl of the nucleoside joined to a polymer support. The product of this reaction is the H-phosphonate diester (21) .
Unlike in the phosphite triester chemistry, this linkage (22) is stable to the acidic conditions (3% trichloroacetic acid in dichloromethane) required for removal of the 5'dimethoxytrityl group. Thus, it is unnecessary to carry out oxidation at every cycle.
Chain elongation therefore consists of only two steps. Oxidation to the phosphodiester with aqueous iodine is completed following oligonucleotide synthesis.
Alternatively oxidation can be carried out under non-aqueous conditions using various reagents which has enabled the synthesis of a number of modified oligonucleotides, a.o. phosphoramidates, phosphorothioates and phosphoro-selenoates.
After the oxidation step, aqueous ammonia is used to remove nucleobase amide protecting groups and to cleave the oligonucleotide from the support.

Result of an oligonucleotide synthesis :
- single-stranded fragments : but double-stranded if perfect palindromic oligonucleotides
(or partially double-stranded if partially palindromic ; may contain a loop)
fragments have 5’- and 3’-OH : ligation requires a 5’-phosphate and 3’-OH
- sizes from n=2 to at least 250 are feasible; but standard sizes are rather from 15 to about 100
Oligonucleotides with variations in the phosphate group : (chirality ! ? !)
- phosphorothioates (1 S instead of O in phosphate)
phosphorodithioates (both O’s replaced by S' : one P-S- + one P=S)
- methylphosphonates (CH
3 instead of OH)
- phosphate-pentose backbone completely replaceable by e.g. a peptide backbone => PNA ("peptide nucleic acid")
LNA : “locked” nucleic acid : bonds (via O) between C2’ and C4’
=> more rigid structure with particular (and better) hybridisation properties

N
N
N
Modified oligonucleotides modified bases: hypoxanthine
2,6-diaminopurine
5-methyl-cytosine
N,N-ethanocytosine
Some of the many different possibilities of synthetic (chemically-modified) oligonucleotides

PNA (peptide nucleic acid)
Consists of repeating units of
N-(2-aminoethyl)-glycine units linked by amide bonds.
Unlike the natural DNA backbone, no deoxyribose or sugar groups are present. The bases are attached by methylene carbonyl linkages.
The positions of the bases in space resemble to the ones in a regular nucleotide chain and allow for hybridisation.

PNA
Several variations are possible (e.g. pPNA : N-(2-amino-ethyl)-phosphonoglycine has a negative charge, to improve its water solubility.)
PNAs can bind in antiparallel, but also in parallel orientation ; and may form triple-helix structures.
A
Triple helix formation with PNA (-derivatives) : two extra chains (one of which is in parallel orientation) (fig A) or with a so-called “clamp” (fig B)
B

PNA (peptide nucleic acid)
The lack of electrostatic repulsion between the two strands in a PNA/nucleic acid duplex leaves the T m largely independent of salt concentration (Figure) .
This allows binding, in the absence of salt, of a PNA probe to a nucleic acid target despite the presence of competing secondary structure. It is noticed from the curve that there is a significant difference at physiological salt, and that the T m s are essentially the same at sodium ion concentrations higher than 500 millimolar.
The T m for PNA-DNA duplexes does not follow the same rules as for DNA/DNA duplexes.
The neutral backbone also increases the rate of hybridization significantly in assays where either the target or the probe is immobilized.
Ionic strength dependence of thermal stability (T m
) of
PNA/DNA (open o) and DNA/DNA duplex (filled o).

LNA
( BNA : "bridged nucleic acid"
ENA : "ethylene-bridged nucleic acid"
The pentose part is not planar : the C
3’
- C
2’ bond is inclined against the planar
C
4’
-O-C
1’
. This can be in 2 ways (with C
2
’ closer to the C
5
’
(named C
2'
-endo) or with the C
3’
(C
3'
-endo) ). (Also sometimes indicated as North for C
3'
-endo)
The position can be fixed by an extra binding (by an oxygen, or a methylene bridge, or larger) between C
2’ and C
4’
.

Bridged nucleic acids ( BNAs ) are modified RNA nucleotides. They are sometimes also referred to as constrained or inaccessible RNA molecules.
BNA monomers can contain a five-membered, six-membered or even a seven-membered bridged structure with a “fixed” C
3
’-endo sugar puckering. The bridge is synthetically incorporated at the 2’, 4’-position of the ribose to afford a 2’, 4’-BNA monomer. The monomers can be incorporated into oligonucleotide polymeric structures using standard phosphoramidite chemistry.
BNAs are structurally rigid oligonucleotides with increased binding affinities and stability.
The nature of the bridge can vary for different types of monomers. The 3D structures for A-
RNA and B-DNA were used as a template for the design of the BNA monomers. The goal for the design was to find derivatives that possess high binding affinities with complementary RNA and/or DNA strands.

The presence of 2’-hydroxyls in the RNA backbone favors a structure that resembles the
A-form structure of DNA. The flexible five-membered furanose ring in nucleotides exists in equilibrium of two preferred conformations of the
N- (C
3
’-endo, A-form) and the
S-type (C
2
’-endo, B-form).
An increased conformational inflexibility of the sugar moiety in nucleosides
(oligonucleotides) results in a gain of high binding affinity with complementary single-stranded RNA and/or double-stranded DNA.
The first 2’,4’-BNA (LNA) monomers were first synthesized in 1997 (T.Imanishi’s group) and in 1998 (J.Wengel’s group.

5'
4'
3'
2'
1'
Chemical structure of an LNA monomer : an additional bridge bonds the 2' oxygen and the 4' carbon of the pentose
5'
4'
3'
2'
1' Conformational structure of an LNA monomer β-D configuration

LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. Such oligomers are synthesized chemically and are commercially available.
The locked ribose conformation enhances base stacking and backbone pre-organization.
This significantly increases the hybridization properties (T m
) of oligonucleotides.

LNA oligonucleotides have specific, useful characteristics .
Some major benefits are :
- increased thermal stability of duplexes as probes in hybridisation
- capable of single nucleotide discrimination
- facilitates T m normalization
- compatible with standard enzymatic reactions, but resistance to exo- and endonucleases ( in vitro and in vivo )
- particularly useful for the detection of short RNA and DNA targets
- strand invasion properties enables detection of “hard to access” samples
- growing list of diagnostic, as well as therapeutic applications
Major drawback/limitations : exclusive rights to LNA technology by Exiqon A/S, and for therapeutic uses exclusive rights to Santaris Pharma (both Danish companies)

:
Requirements that may apply, depending on specific applications or situations :
The label should be easy to attach under mild conditions using a simple, cheap and reproducible protocol.
The method of attachment should not interfere with the signal-generating properties of the label or affect the hybridization of the oligonucleotide/DNA to its target nucleic acid.
The label should be detectable at low concentrations (using suitable instrumentation).
In some cases it may be advantageous to add multiple labels to a single oligonucleotide ; the label should be compatible with this.
The label should be stable to hybridization conditions e.g. elevated temperatures (up to
96 °C), detergents, and aqueous buffers up to pH 9.
The label should be stable to long term storage at −20 °C and be easily disposable.
It should be possible to simultaneously detect several different labels simultaneously and to differentiate between them (multiplex detection).

The architecture of a label

Oligonucleotides can be synthesized having a 5' amino group, a 5' phosphate, a 5' thiol (-SH), and many others.
These are targets for addition of a signaling moiety by specific chemistries, depending on the reactive group of the label and the nature of the modified 5'-end of the oligonucleotide.
Similarly, additions to the 3'-end, and at internal positions are also possible.
Below some examples are shown, but many more possibilities exist.
- direct incorporation of biotin at internal positions or at the 5' or the 3' end
=> similarly for incorporation of fluorescent (and other) groups
- incorporation of - modified bases, e.g. hypoxanthine (nucleoside = inosine)
- modified phosphates
- synthesis of a -anomeres
- oligonucleotides with a reactive NH
2 group at the 5’-end, enabling covalent coupling
(typically as succinimide ester) of other components such as biotin, digoxygenin, fluorescein, enzymes, etc. Also with SH as 5'-reactive target.

Examples of commonly used reactions to attach a label.
(L = label + spacer)

Chemical synthesis of oligonucleotides with a 5' or 3' terminal phosphate
5'
A special starting block is used, followed by addition of the
"phosphate monomer" and then continuing the regular synthesis.
At the time of deprotection with
NH
3
, cleavage into three products occurs as shown in the figure.
A phosphate residue remains at the 3'-end.
3'

Oligonucleotide with a
5' thiol. (thiohexyl extension)

Enzyme labeling

Internal, or terminal incorporation of biotin at either the 5'- or the 3'end. a protecting group at the biotin moiety biotin to 5’ end internal incorporation of biotin in a growing oligonucleotide chain biotin to 5’ end biotin to 3’ end

Incorporation of fluorescein at the 5'-end or 3'-end of a synthetic oligonucleotide.

Using aminolink to prepare oligonucleotides bearing a 5' amino-group. Tetrazole activates the aminolink in the coupling reaction.
Numerous 'labels', e.g. dyes, can be attached, linking biotin to 5’ amino group

Enzymatic incorporation of biotin or digoxigenin by polymerases and modified dNTP's.
Similarly, other modifications can be introduced.

Applications of fluorescent oligonucleotide analogs :
Use of FRET : (fluorescence resonance energy transfer).
- distance between the donor-acceptor pair of primary importance :
==> fluorescence of Edans (493 nm) quenched by Dabcyl
In the Taqman system (for qPCR)
- quenching upon hybridisation
- exonucleolytic removal of fluorescent group leads to fluorescence.
"Molecular beaken"
- for detection in hybridisation experiments: fluorophor e + quencher at termini
- stem-loop ==> quenching
- hybridisation disrupts the stem ==> activation
Scorpion primers : internal loop complementary to target (first order renaturation)
ET-primers (Energy Transfer) (for DNA sequencing) (cfr. Bigdyes)

FRET : f luorescence ( F örster) r esonance e nergy t ransfer
A donor chromophore in its excited state can transfer energy by a nonradiative, long-range dipole -dipole coupling mechanism to an acceptor chromophore in close proximity (typically <10nm).

Molecular beacon
Quenching of fluorescence if the stem-loop structure is kept together.
Binding of the target disrupts the short helix and separates fluorophore from quencher, thus emitting fluorescence.
second-order kinetics

Real-time quantitative PCR
Liberation of “reporter” is proportional to the quantity of produced amplicon DNA.
The use of a set of different fluorophores (different colors) enables testing different targets in parallel (if they display the same efficiency of amplification).

Use of
Scorpion probes
Intramolecular hybridisation of the extended sequence disrupts the quenching of the fluorescence. first-order kinetics
(unimolecular reaction)
(much faster than with a molecular beacon)

PCR blocker : often a hexethylene glycol modification linker sequence
T-BHQ
®
: thymidine labeled with a "black hole quencher" if loop present => FRET quenching
3'-end

Activated fluorescent dyes for attachment to oligonucleotides in preparation of ET-primers

Synthesis of ”larger” fragments by stepwise assemby ; may also be done in combination with filling-in reactions using DNA polymerase :
Ligation strategie for complete gene synthesis according to G. Khorana.
- stepwise assemby, requiring selective phosphorylation of the segments to ligate
- overlapping sections between the strands must be sufficiently unique to deliver exclusively the aimed-at combinations.
(ps. too long strands may hybridize too slowly !!!)

Combinations using enzymatical synthesis (filling-in reactions by DNA polymerase) completely chemical synthesis (if ligated) or filling-in reaction of one strand filling-invul reaction on both strands
(overlapping 3’ ends)
- after enzymatical filling-in, the resulting ends always are blunt , and lack the phosphate moiety
a restriction recognition sequence may allow to adapt those (and yield a 5’ terminal phosphate)

Nomenclature :
- linker, adaptor, connector
- primer, degenerated primer
- probe, mixed probe, degenerated probe relation between amino-acid sequence <> nucleotide sequence e.g.
M - A - W - R - Y - T -
5’
N-
4 2 4 2 (4)
= 64 oligonucleotides the 17-mer mixture represents all the potential coding sequences
(and more) for that particular peptide sequence remark : not all 64 oligomers encode the exact sequence
(see arginine triplet, etc.)

Photolithografic DNA synthesis : parallel large-scale synthesis
- for large-scale synthesis of oligonucleotides in oligonucleotide arrays (' DNA-chips ') :
- light-directed synthesis (adopted from the semiconductor industry) : selective removal of 5'- (or 3'-) protecting groups on a glass support by controlled exposure to light (365 nm) through a photolithografic mask. Only the positions that are illuminated (i.o.w. that have a free OH-group) participate in the subsequent coupling reaction. This allows parallell synthesis of thousands of oligonucleotides.
- Photolabile protecting group => activation with light => reaction with the next nucleotide (using standard phosphoramidite chemistry) :
- cycles of photodeprotection and nucleotide addition : e.g. for an array of 100 or 1000 or more decamers :
- only 40 cycli required (i.e. 10 for each of the 4 nucleotides, cfr. pyrosequencing).
- Terminology :
WAFER : the disk onto which the array is made
FEATURE : one element in the array features smaller than 24x24 m m can be made => 1,6 cm 2 chip features of 10x10 m m are being attempted => more than 10 6 sequences/chip

The photodeprotection reaction (photolysis reaction of MeNPOC protecting group) is done at 365 nm (I-line of an Hg lamp) (near UV because at wavelengths below 360 nm modifications at the nucleotide bases are likely) .
Complete deprotection in less than 1 minute.
Efficiencies of about 90-95% per step are attainable : for a 20-mer synthesis this means that only about 10% of the total end product will be the particular oligonucleotide sequence.
For hybridisation purposes this is acceptable (although there will be a substantial background) (see elsewhere : sequence analysis by SBH) .

In another approach, the removal of the 5'O -DMT group is effected by electrochemical generation of the acid at the required site(s) and cleavage results in a few seconds.
The maximum number of different oligomers is 4 n
(n = size of the sequence) e.g. 4 6 equals 4096 hexamers, 4 8 = 65,536 octamers,
4 10 = more than 1 million decamers (1,048,576)
==> all 16-mers = 4,294,967,296) with nanotechnology this comes (or is) within reach.
(the future will show how far we can go).
But never forget that resolving longer homopolymeric stretches requires extra size delimiters.